Thermostable esterase and its gene

ABSTRACT

The present invention relates to an esterase having the excellent thermostable property which can be utilized for ester hydrolysis reaction, ester synthesis reaction, ester interchange reaction and the like and its gene.  
     The present invention provides  
     1. an esterase which is characterized in that it has at least a partial amino acid sequence necessary for expressing the thermostable esterase activity among the amino acid sequence shown by SEQ ID: No.1 having any one of the following amino acid substitutions:  
     (1) amino acid substitution where 325th amino acid in the amino acid sequence shown by SEQ ID: No.1 is substituted with isoleucine,  
     (2) amino acid substitution where 240th amino acid in the amino acid sequence shown by SEQ ID: No.1 is substituted with alanine, and 288th amino acid is substituted with alanine,  
     (3) amino acid substitution where 43rd amino acid in the amino acid sequence shown by SEQ ID: No.1 is substituted with serine,  
     2. a gene which is characterized in that it encodes the above esterase,  
     3. a plasmid which is characterized in that it contains the above gene,  
     4. a microorganism which is characterized in that it contains the above plasmid, and  
     5. a process for producing an esterase, which is characterized by comprising culturing the above microorganism and, thereby, allowing the microorganism to produce an esterase having at least a partial amino acid sequence necessary for expressing the thermostable esterase activity among the amino acid sequence shown by SEQ ID: No.1 having any one of the following amino acid substitutions:  
     (1) amino acid substitution where 325th amino acid in the amino acid sequence shown by SEQ ID: No.1 is substituted with isoleucine,  
     (2) amino acid substitution where 240th amino acid in the amino acid sequence shown by SEQ ID: No.1 is substituted with alanine and 288th amino acid is substituted with alanine,  
     (3) amino acid substitution where 43rd amino acid in the amino acid sequence shown by SEQ ID: No.1 is substituted with serine.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to an esterase having the excellentthermostable property which can be utilized for ester hydrolysisreaction, ester synthesis reaction, ester interchange reaction and thelike and its gene.

[0003] 2. Description of the Related Art

[0004] Esterase is an enzyme which hydrolyzes an ester linkage and hasability to catalyze ester synthesis and ester interchange reaction, andhas been recently utilized in organic synthesis reaction formanufacturing medicaments, pesticides or intermediates thereof.

[0005] It is desirable that the esterase, which is industriallyutilized, has high stability to temperature, pH, solvent, pressure andthe like. Inter alia, where the esterase has high thermostability, thereaction temperature can be elevated, enabling the reaction rate to beenhanced and an inactivation of the enzyme to be reduced. Accordingly,there is desired the esterase having the excellent thermostability forshortening the reaction time and promoting the reaction efficiency.

DISCLOSURE OF THE INVENTION

[0006] Under these circumstances, the present inventors studied hardusing the technique of introducing mutation into gene by site-directedmutagenesis and, as a result, found that mutant esterase having theamino acid sequence where the particular amino acid in the wild-typeamino acid sequence is substituted shows the excellent thermostability,which resulted in completion of the present invention.

[0007] That is, the present invention provides:

[0008] 1. an esterase (hereinafter referred to as “the presentesterase”) which is characterized in that it has at least a partialamino acid sequence necessary for expressing the thermostable esteraseactivity among the amino acid sequence shown by SEQ ID: No.1 having anyone of the following amino acid substitutions:

[0009] (1) amino acid substitution where 325th amino acid in the aminoacid sequence shown by SEQ ID: No.1 is substituted with isoleucine,

[0010] (2) amino acid substitution where 240th amino acid in the aminoacid sequence shown by SEQ ID: No.1 is substituted with alanine and288th amino acid is substituted with alanine,

[0011] (3) amino acid substitution where 43rd amino acid in the aminoacid sequence shown by SEQ ID: No.1 is substituted with serine,

[0012] 2. an esterase which is characterized in that it has at least apartial amino acid sequence necessary for expressing the thermostableesterase activity among the amino acid sequence shown by SEQ ID: No.1having amino acid substitution where 325th amino acid in the amino acidsequence shown by SEQ ID: No.1 is substituted with isoleucine,

[0013] 3. an esterase which is characterized in that it has at least apartial amino acid sequence necessary for expressing the thermostableesterase activity among the amino acid sequence shown by SEQ ID: No.1having amino acid substitution where 240th amino acid in the amino acidsequence shown by SEQ ID: No. 1 is substituted with alanine, and 288thamino acid is substituted with alanine,

[0014] 4. an esterase which is characterized in that it has at least apartial amino acid sequence necessary for expressing the thermostableesterase activity among the amino acid sequence shown by SEQ ID: No.1having amino acid substitution where 43rd amino acid in the amino acidsequence shown by SEQ ID: No.1 is substituted with serine,

[0015] 5. a gene which is characterized in that it encodes the esteraseof the above 1 to 4,

[0016] 6. a plasmid which is characterized in that it contains the geneof the above 5,

[0017] 7. a microorganism which is characterized in that it contains theplasmid of the above 6,

[0018] 8. a process for producing an esterase which is characterized bycomprising culturing the microorganism of the above 4 and, thereby,allowing the microorganism to produce an esterase having at least apartial amino acid sequence necessary for expressing the thermostableesterase activity among the amino acid sequence shown by SEQ ID: No.1having any one of the following amino acid substitutions:

[0019] (1) amino acid substitution where 325th amino acid in the aminoacid sequence shown by SEQ ID: No.1 is substituted with isoleucine,

[0020] (2) amino acid substitution where 240th amino acid in the aminoacid sequence shown by SEQ ID: No.1 is substituted with alanine and288th amino acid is substituted with alanine,

[0021] (3) amino acid substitution where 43rd amino acid in the aminoacid sequence shown by SEQ ID: No.1 is substituted with serine.

[0022] Further scope of applicability of the present invention willbecome apparent from the detailed description given hereinafter.However, it should be understood that the detailed description andspecific examples, while indicating preferred embodiments of theinvention, are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

[0023] Throughout this specification and the claims which follow, unlessthe context requires otherwise, the word “comprise”, and variations suchas “comprises” and “comprising”, will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integer orstep.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024]FIG. 1 is a view showing a restriction enzyme map of a plasmidpCC6 containing a gene encoding a wild-type esterase.

[0025]FIG. 2 is a view showing a step of constructing the expressionplasmid pCC101 containing a gene encoding the wild-type esterase.

[0026]FIG. 3 is a view showing a restriction enzyme map of an expressionplasmid pCC101 containing a gene encoding the wild-type esterase. In thefigure, an open symbol indicates a DNA derived from ChromobacteriumSC-YM-1 (FERM BP-6703) and a black part indicates the translation regionof the wild-type esterase.

[0027]FIG. 4 is a view showing a base sequence of a syntheticoligonucleotide used for introducing a site-directed mutation into 43rdamino acid, 240th amino acid, 288th amino acid, 325th amino acid and363rd amino acid of the wild-type esterase.

[0028]FIG. 5 is a view showing a step for constructing the plasmidpCCN43S containing the present gene.

[0029]FIG. 6 is a view showing a step for constructing the plasmidpCCT240A containing the present gene.

[0030]FIG. 7 is a view showing a step for constructing the plasmidpCCV288A containing the present gene.

[0031]FIG. 8 is a view showing a step for constructing the plasmidpCCV325I containing the present gene.

[0032]FIG. 9 is a view showing a step for constructing the plasmidpCCA363term containing the present gene.

[0033]FIG. 10 is a view showing a step for constructing the plasmidpCCN43SA363term containing the present gene.

[0034]FIG. 11 is a view showing a step for constructing the plasmidpCCT240V288A containing the present gene.

DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention will be described in detail below.

[0036] Esterase having the amino acid sequence shown by SEQ ID: No.1(hereinafter referred to as “wild-type esterase”) is an esterasedescribed in JP-A-5-315497. The esterase activity of the esterase or thepresent esterase can be determined by mixing with, for example,p-nitrophenyl acetate (pNPA), holding a temperature at 37° C. andquantitating the amount of released p-nitrophenyl using absorbance ofthe reaction solution at 410 nm. In the present esterase, “thethermostable esterase activity” means that the remaining activitypercentage is, for instance, high as compared with the wild-typeesterase even after holding a temperature at 70° C. for 120 minutes.

[0037] In addition, in the present esterase, “at leas, a partial aminoacid sequence necessary for expressing the thermostable esteraseactivity” is, for example, an esterase comprising 362 amino acidscorresponding to at lease 1st to 362nd amino acids in the amino acidsequence shown by SEQ ID: No.1, and its equivalents which have the samebiological function.

[0038] In order to obtain a gene (hereinafter referred to as “thepresent gene”) which is characterized in that it encodes the presentesterase, a gene encoding the wild-type esterase (hereinafter referredto as “wild-type gene”) may be firstly obtained. The wild-type gene is,for example, a gene having the base sequence shown by SEQ ID: No.2 andmay be obtained from microorganisms belonging to genus Chromobacteriumretained by a microorganism retaining organization and the likeaccording to the conventional genetic engineering technique describedin, for example, J. Sambrook, E. F. Fritsch, T. Maniatis; MolecularCloning 2nd edition, published by Cold Spring Harbor Laboratory, 1989.That is, a microorganism belonging to genus Chromobacterium is culturedusing, for example, LB medium (tryptophane 1.0%, yeast extract 0.5%,NaCl 0.5%), the cells of the microorganism obtained by culturing aredisrupted according to the conventional method such as ultrasonicdisruption and the like, treated with protease, and genomic DNA isextracted. The resulting genomic DNA is cleaved with a suitablerestriction enzyme, and inserted into λ gtII which is a phage vector orpUC19 which is a plasmid vector and the like using a ligase to make agenomic DNA library. This can be screened with, for example, a screeningmethod such as hybridization method using a synthetic DNA probecorresponding to the portion of the amino acid sequence of the wild-typeesterase, a method for measuring the activity of the wild-type esteraseand the like, to obtain a clone containing the wild-type gene. As asynthetic DNA probe corresponding to the portion of the amino acidsequence of the wild-type esterase, in particularly, for example, anoligonucleotide having the base sequence shown by SEQ ID:No.3 and anoligonucleotide having the base sequence shown by SEQ ID: No.4 may beused.

[0039] The present gene may be prepared by introducing a site-directedmutation into the wild-type gene. As a site-directed mutationintroducing method, there are, for example, a method by Olfert Landt etal. (Gene, 96, 125-128, 1990), a method by Smith et al. (GeneticEngineering, 31, Setlow, J. and Hollaender, A. Plenum:New York), amethod by Vlasuk et al. (Experimental Manipulation of Gene Expression,Inouye, M.: Academic Press, New York), a method by Hos.N.Hunt et al.(Gene, 77, 51, 1989) and the like.

[0040] For example, in order to prepare the present gene where 325thamino acid in the amino acid sequence shown by SEQ ID: No.1 issubstituted with isoleucine, a plasmid DNA which contains the wild-typegene having a base sequence shown by SEQ ID:No.2 is first preparedaccording to the method described in, for example, J. Sambrook, E. F.Fritsch, T. Maniatis; Molecular Cloning 2nd edition, published by ColdSpring Harbor Laboratory, 1989 and the like. Then, by using the plasmidDNA as a template, and by using as one side primer an oligonucleotidecomprising a base sequence corresponding to the amino acid sequencewhere 325th amino acid in the amino acid sequence shown by SEQ ID: No.1is substituted with isoleucine (for example, oligonucleotide having abase sequence shown by SEQ ID: No. 15) and as the other side primer anoligonucleotide having a base sequence shown by SEQ ID: No.20,amplification may be performed by a PCR method. Here, PCR reactionconditions are as follows: after maintaing a temperature at 94° C. for 5minutes, 20 cycles of treatment of maintaining a temperature at 94° C.for 1 minute, then at 50° C. for 2 minutes and at 75° C. for 3 minutesare carried out and finally a temperature is maintained at 75° C. for 8minutes. The DNA fragment thus amplified may be digested with, forexample, restriction enzyme BstPI and XbaI, and ligation-reacted withthe plasmid DNA comprising the wild-type esterase gene same digestedwith the same restriction enzyme to obtain the desired present gene.

[0041] In addition, an oligonucleotide comprising a base sequencecorresponding to the amino acid sequence where 240th amino acid in theamino acid sequence shown by SEQ ID: No.1 is substituted with alanineand 288th amino acid is substituted with alanine and an oligonucleotidecomprising a base sequence corresponding to the amino acid sequencewhere 43rd amino acid in the amino acid sequence shown by SEQ ID: No. 1is substituted with serine may be prepared using a similar method tothat described above. The detail thereof is described as Examples.

[0042] By using the present gene thus prepared, the present esterase maybe produced and obtained at a large amount according to the conventionalgenetic engineering method. More particularly, for example, a plasmidwhich can express the present gene in the host microorganism isprepared, which may be introduced into the host microorganism totransform to make a transformant microorganism. Then, the resultanttransformant microorganism may be cultured according to the conventionalmicroorganism culturing method.

[0043] The example of the above plasmid are those that can be replicatedin the host microorganism and is easily isolated and purified from thehost microorganism. Preferably, mention may be made of a plasmid wherethe present gene is introduced into an expression vector having apromoter and a detectable marker. As an expression vector, variouscommercially available vectors may be used. For example, in the case ofexpression in E. coli, an expression vector comprising a promoter suchas lac, trp, tac and the like (manufactured by Pharmacia Biotech and thelike) may be used.

[0044] As a host microorganism, both eukaryote and prokaryote can beused and an example thereof is E. coli and the like. The above plasmidmay be introduced into the host microorganism by the conventionalgenetic engineering method to transform the host microorganism.

[0045] Culturing of the microorganism (hereinafter referred to as thepresent microorganism) harboring the plasmid containing the present genethus obtained may be performed according to the conventionalmicroorganism culturing method. For example, where the hostmicroorganism is E. coli, culturing is performed in a mediumappropriately containing a suitable carbon source, a nitrogen source anda minor nutrient such as vitamines. As a culturing method, both solidculturing and liquid culturing are possible and, preferably, mention maybe made of an aerated stirring culturing method.

[0046] The present microorganism producing the present esterase thusprepared may be utilized for producing a useful compound such asmedicaments, pesticides, and intermediates thereof and the like as abioreactor for ester hydrolyzation, ester synthesization, esterinterchage reaction or the like.

[0047] In addition, from the cells obtained by culturing the presentmicroorganism, an extract containing the present esterase may beprepared or the present esterase may be collected and purified and thesemay be utilized as an enzyme reactor. Collection and purification ofesterase from the cells obtained by culturing the present microorganismmay be performed by suitably combining the conventional proteinextracting, isolating and purifying methods. For example, aftercompletion of the culturing, the cells of the present microorganism arecollected by centrifugation or the like, disrupted or lyzed and thepresent esterase may be collected and purified by combining the stepsusing various chromatographies such as ion exchange, hydrophobicity, gelfiltration and the like.

[0048] The present microorganism and the present esterase describedabove may be utilized as a reactor by immobilizing onto a suitablecarrier.

EXAMPLE

[0049] The following Examples illustrate the present invention in detailbut the present invention is not limited to them.

Example 1 Preparation of Wild-type Gene: Preparation of Genomic DNA

[0050] Chromobacterium strain SC-YM-1 (this strain was originallydeposited in National Institute of Bioscience and Human-TechnologyAgency of Industrial Science and Techonology as an asccession No. FERMP-14009 by the applicant on Dec. 9, 1993 and at present continuouslydeposited as an accession No. FERMBP-6703 under Budapest Treaty) wascultured by shaking in 5 ml of a medium for pre-culturing (glucose 1%(w/v), yeast extract 1% (w/v), K₂HPO₄ 0.1% (w/v), MgSO₄ 0.02% (w/v),pH7.3) at 30° C. for 24 hours and the resulting culture solution wasinoculated on 1000 ml of medium for culturing (glucose 1% (w/v), yeastextract 1% (w/v), K₂KPO₄ 0.1% (w/v), MgSO₄ 0.02% (w/v), pH 7.3),followed by culturing at 30° C. Upon this, when OD₆₆₀ reached 3.4,penicillin G was added to the final concentration of 2 units/ml culturesolution and culturing was continued until OD₆₆₀ reached 10.

[0051] The cells were collected by centrifugation (8000×g, 10 min., 4°C.), the cells were suspended in 80 ml of 10 mM Tris buffer (pH 8.0),25% (w/v) sucrose solution, and to this was Lysozyme egg white(manufactured by Seikagaku corporation) to the final concentration of 5mg/ml, followed by incubation at 37° C. for 30 minutes. Then, 10 ml of10% (w/v) SDS was added and protease K (manufactured by Boehringer) wasadded to the final concentration of 200 μg/ml, followed by incubation at37° C. for 3 hours. Thereafter, extraction was performed with anequivalent volume of 0.1M Tris-saturated phenol three times and withether two times, and 2 volumes of ethanol was added to the aqueous layerto stir, followed by centrifugation (12000×g, 30 min., 4° C.). Theresulting precipitates were dried and dissolved in 20 ml of Tris EDTAbuffer (10 mM Tris-HCl, 1mM EDTA, pH 8.0), and subjected to CsCl-EtBrequilibrium density-gradient ultracentrifugation (275000×g, 18 hours,2520 C.) to recover the band-likely converged DNA which was dialyzedagainst Tris EDTA buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to obtainabout 5.4 mg of genomic DNA.

Example 2 Preparation of Wild-type Gene: Preparation of Genomic DNALibrary

[0052] 100 μg of the genomic DNA obtained in Example 1 was digested withXhoI (manufactured by Takara Shuzo Co., Ltd.). On the other hand, 1 μgof λ phage λ ZAPII (manufactured by Stratagene) was digested with XhoI,mixed with the genomic DNA digests, and a ligase (manufactured by TakaraShuzo Co., Ltd.) was added to maintain at 16° C. overnight.

[0053] Then, the DNA contained in this reaction solution was packed intoλ phage A ZAPII using an in vitro packaging kit (manufactured byStratagene) and E. coli strain XL-1blue to make the genomic DNA library.

Example 3 Preparation of Wild-type Gene: Screening of Genomic DNALibrary

[0054] 1. Preparation of Synthetic DNA Probe and Labeling with Isotope

[0055] 44-mer oligonucleotides having a base sequences shown by SEQ ID:Nos. 3 and 4 were synthesized based on the amino acid sequence of theN-terminal of the wild-type esterase. The synthesis of theoligonucleotides was performed using a DNA synthesizer (AppliedBiosystems Model 394A).

[0056] Into 50 pmol of this oligonucleotide were mixed 3 μl of 0.5MTris-HCl (pH 7.6), 0.1M MgCl₂, 0.05M DTT, 0.001M EDTA, 10 units of T4Polynucleotide Kines (manufactured by Takara Shuzo Co., Ltd.) and 10 μlof [γ ³²P]ATP (manufactured by Amersham), which was maintained at 37° C.for 60 minutes, and subjected to gel filtration by Sephadex G-50(manufactured by Pharmacia) to make a DNA probe labeled with an isotope.

[0057] 2. Screening of Genomic DNA Library

[0058]E. coli infected with the phage of the genomic DNA library made inExample 2 was spread on a plate to culture, a nitrocellulose filter wastightly contacted on the surface of the plate and mildly peeled. Thefilter was soaked into 1.5 M NaCl-0.5 M NaOH solution, and then soakedinto 1.5 M NaCl-0.5 M Tris-HCl (pH 8.0) solution to neutralize.Thereafter, the filter was washed with 0.36 M NaCl-20 mM NaH₂PO₄ (pH7.5)-2 mM EDTA (pH 7.5) and then dried.

[0059] Then, by using the filter and the isotope-labeled DNA probecorresponding to the N-terminal amino acid sequence of the wild-typeesterase prepared above, plaque hybridization was performed by thefollowing method. That is, the filter was maintained at 60° C. for 30minutes in a solution containing 4×SSC, 1% (w/v) SDS, 10×Dendhart (0.2%(w/v) Ficoll, 0.2% (w/v) polyvinyl pyrrolidone and 0.2% (w/v) bovineserum albumin), and maintained at 60° C. for 5 hours in a solutioncontaining 5×SSC, 5×Dendhart and 100 μg/ml salmon sperm DNA.Hybridization was performed by placing a solution containing 5×SSC,5×Dendhart and 100 μg/ml salmon sperm DNA and the filter in a plasticbag, adding the isotope-labeled DNA probe at about 5×105 cpm per filterand maintaining at 60° C. overnight.

[0060] The filter by which hybridization was performed as above waswashed by successively maintaining 1) at 60° C. for 15 minutes in asolution containing 2×SSC and 0.5% (w/v) SDS, 2) at 25° C. for 30minutes in a solution containing 2×SSC and 0.5% (w/v) SDS, 3) at 60° C.for 15 minutes in a solution containing 2×SSC and 0.5% SDS(w/v),air-dried and autoradiographied by contacting with a X-ray film (FUJIRX) and an intensifying paper at −80° C. overnight. As a result, aplaque giving a positive signal was obtained. The desired plaque, helperphage and E. coli were mixed, cultured at 37° C. for 4 hours andmaintained at 70° C. for 20 minutes according to the conventional methoddescribed by authors, J. Sambrook, E. F. Fritsch, T. Maniatis; MolecularCloning 2nd edition, published by Cold Spring Harbor Laboratory, 1989.E. coli was infected with the supernatant and cultured to obtain atransformant growing in a medium containing ampicillin. A plasmid DNAwas prepared from the resultant transformant, the base sequence thereofwas determined by a dideoxy method and, as a result, it was found thatthe resultant clone did not encode the full length of an esterase gene.Then, by using a DNA fragment having a portion of the sequence as aprobe, screening by plaque hybridization was performed again. Upon this,a libraty was used which was made by partially digesting the genomic DNAwith Sau3AI (manufactured by Takara Shuzo Co., Ltd.) and ligating to λphage λ ZAPII (manufactured by Stratagene). As a result, a plaque givinga positive signal was obtained. A plasmid DNA was prepared from theplaque according to the same method as that described above, the basesequence was determined and, as a result, it was found that it encodedthe full length of an esterase gene. Thus, the plasmid pCC6 (FIG. 1) wasobtained.

Example 4 Expression Plasmid Containing Wild-type Gene

[0061] In order to convert the base sequence around an initiation codonof the wild-type gene and that 5′ upstream thereof into a sequencesuitable for gene expression in E. coli, oligonucleotides having a basesequences shown by SEQ ID: Nos.5-11 were synthesized using a DNAsynthesizer Model 394A (manufactured by Applied Biosystems).

[0062] LP-1 (SEQ ID: No.5)

[0063] LP-2 (DEQ ID: No.6)

[0064] ES-3 (DEQ ID: No.7)

[0065] ES-4 (SEQ ID: No.8)

[0066] ES-5 (SEQ ID: No.9)

[0067] ES-6 (SEQ ID: No.10)

[0068] ES-7 (SEQ ID: No.11)

[0069] The 5′ terminals of oligonucleotides LP-2, ES-3, ES-5, ES-6 andES-7 were phosphorylated, ligated with LP-1 and ES-5, and annealed toprepare a double-stranded DNA fragment (SD) comprising the followingbase sequence. The double-stranded DNA fragment (SD) was phosphorylatedat its both ends. (SD) <    LP-1      ><        ES-7             ><AATTCTTTTT TAATAAAATC AGGAGGAAAA AACATATGAC TCTGTTCGAT GGTATCACTT   GAAAAA ATTATTTTAG TCCTCCTTTT TTGTATACTG AGACAAGCTA CCATAGTGAA   <          LP-2             ><            ES-6 EcoRI     ES-3      ><        ES-5            > CGCGAATCGT AGATACTGATCGTCTGACTG TTAACATCCT GGAACGTGC GCGCTTAGCA TCTATGACTA GCAGACTGACAATTGTAGGA CCTTGCACGC CGG     ><                        ES-4                  >                                                Eco52I

[0070] On one hand, the Sac I fragment (about 3.5 kbp) in pCC6 wassubcloned into pUC118 (manufactured by Takara Shuzo Co., Ltd.) to makepCC30. This pCC30 was digested with Eco52I and SacI to excise a DNAfragment (about 1.2 Kbp) encoding the translation region of an esterasegene. On the other hand, pUC118 having the lac promoter was digestedwith EcoRI and SacI and treated with alkaline phosphatase. TheEco52I-SacI fragment containing the above DNA fragment (SD) and thetranslation region of the present gene was ligated to the region betweenthe EcoRI site and the SacI site of this pUC118 using a DNA ligation kit(manufactured by Takara Shuzo Co., Ltd.) (FIG. 2) to make pCC101 (FIG.3).

Example 5 Preparation of the Present Gene

[0071] 1. Preparation of Mutant Primer

[0072] As a mutant primer for introducing an amino acid substitutionAsn43Ser (amino acid substitution of Asn of 43rd amino acid with Ser),Thr240Ala (amino acid substitution of Thr of 240th amino acid with Ala),Val288Ala (amino acid substitution of Val of 288th amino acid with Ala),Val325IIe (amino acid substitution of Val of 325th amino acid with IIe)or Ala363Term (termination codon) (substitution of Ala of 363rd aminoacid with the base sequence showing termination codon), syntheticoligonucleotides (mutant primers N43S, T240A, V288A, V3251, A363Term,RV-G, RV-C, RV-D, MY-2, MY-3, MY-6) were prepared having the basesequence corresponding to each amino acid as shown by FIG. 4 and SEQID:Nos.12-22. These mutant primers were synthesized using a DNAsynthesizer Model 394 manufactured by Applied Biosystems and purifiedwith an oligonucleotide purifying cartridge manufactured by the samecompany.

[0073] 2. Introduction of Site-directed Mutation

[0074] A mutant esterase was prepared according to a method of OlfertLandt, et al. (Gene, 96, 125-128, 1990). 2-1) Preparation of pCCN43S(example of substitution regarding 43rd amino acid)

[0075] DNA fragment was amplified with GeneAmpTM PCR Reagent kit(manufactured by Takara Shuzo Co., Ltd.) (1stPCR) using the mutantprimer RV-G (100 pmol) shown by SEQ ID: No.17 and the mutant primer N43S(100 pmol) shown by SEQ ID:No. 12 and using 500 ng of pCCO1I obtained inExample 4 as a template DNA. The resultant PCR product (190 bp fragment)was purified using SUPREC-02 (manufactured by Takara Shuzo Co., Ltd.)column.

[0076] Subsequently, similarly, DNA fragment was amplified withGeneAmpTM PCR Reagent kit using the mutant primer MY-3 (50 pmol) shownby SEQ ID: No.21 and the 190 bp DNA fragment (50 pmol) previouslypurified as a primer and using 500 ng of pCC101 as a template DNA. Theamplified DNA fragment was digested with restriction enzymes NdeI andBpu1102I, the sample was electrophoresed with 4% agarose gel(NuSieve3:1Agarose, manufactured by Takara Shuzo., Co., Ltd.), about 370bp DNA fragments were separated and purified using GeneClean DNApurification kit (manufactured by Bio101).

[0077] On the other hand, 3 μg of pCC101 was digested with NdeI andBpu1102I, and treated with alkaline phosphatase. Then, the NdeI-Bpu1102Ifragment (4.2 Kbp) of this pCC101 and the previously prepared andobtained NdeI-Bpu1102I fragment (240 bp) in which mutation had beenintroduced were ligated using a DNA ligation kit (manufactured by TakaraShuzo Co., Ltd.), and transformed into E. coli strain JM109 according tothe conventional method to make pCCN43S (FIG. 5).

[0078] 2-2) Preparation of pCCT240A (Example of Substitution Regarding240th Amino Acid)

[0079] DNA fragment was amplified with GeneAmpTM PCR Reagent kit(manufactured by Takara Shuzo Co., Ltd.) (1stPCR) using the mutantprimer MY-6 (100 pmol) shown by SEQ ID: No.22 and the mutant primerT240A (100 pmol) shown by SEQ ID:No. 13 and using 500 ng of pCC101obtained in. Example 4 as a template DNA. The resultant PCR product (280bp fragment) was purified using SUPREC-02 (manufactured by Takara ShuzoCo., Ltd.) column.

[0080] Subsequently, similarly, DNA fragment was amplified withGeneAmpTM PCR Reagent kit using the mutant primer RV-C (50 pmol) shownby SEQ ID: No.18 and the 280 bp DNA fragment (50 pmol) previouslypurified as a primer and using 500 ng of pCC101 as a template DNA. Theamplified DNA fragment was digested with restriction enzymes Bpu1102Iand BstPI, the sample was electrophoresed with 4% agarose gel(NuSieve3:1Agarose, manufactured by Takara Shuzo Co., Ltd.), about 590bp DNA fragments were separated and purified using GeneClean DNApurification kit (manufactured by Bio101).

[0081] On the other hand, 3 μg of pCC101 was digested with Bpu1102I andBstPI, and treated with alkaline phosphatase. Then, the Bpu1102I-BstPIfragment (3.8 Kbp) of this pCC101 and the previously prepared andobtained Bpu1102I-BstPI fragment (590 bp) in which mutation had beenintroduced were ligated using a DNA ligation kit (manufactured by TakaraShuzo Co., Ltd.), and transformed into E. coli strain JM109 according tothe conventional method to make pCCT240A (FIG. 6).

[0082] 2-3) Preparation of pCCV288A (Example of Substitution Regarding288th Amino Acid)

[0083] DNA fragment was amplified with GeneAmp TMPCR Reagent kit(manufactured by Takara Shuzo Co., Ltd.) (1stPCR) using the mutantprimer MY-6 (100 pmol) shown by SEQ ID: No.22 and the mutant primerV288A (100 pmol) shown by SEQ ID:No. 14 and using 500 ng of pCC110obtained in Example 4 as a template DNA. The resultant PCR product (130bp fragment) was purified using SUPREC-02 (manufactured by Takara ShuzoCo., Ltd.) column.

[0084] Subsequently, similarly, DNA fragment was amplified withGeneAmpTM PCR Reagent kit using the mutant primer RV-C (50 pmol) shownby SEQ ID: No.18 and the 130 bp DNA fragment (50 pmol) previouslypurified as a primer and using 500 ng of pCC101 as a template DNA. Theamplified DNA fragment was digested with restriction enzymes Bpu1102Iand BstPI, the sample was electrophoresed with 4% agarose gel(NuSieve3:1Agarose, manufactured by Takara Shuzo Co., Ltd.), about 590bp DNA fragments were separated and purified using GeneClean DNApurification kit manufactured by Bio101.

[0085] On the other hand, 3 μg of pCC101 was digested with Bpu1102I andBstPI and treated with an alkaline phosphatase. Then, the Bpu1102I-BstPIfragment (3.8 Kbp) of this pCC101 and the previously prepared andobtained Bpu1102I-BstPI fragment (590 bp) in which mutation had beenintroduced were ligated using a DNA ligation kit (manufactured by TakaraShuzo Co., Ltd.), and transformed into E. coli strain JM109 according tothe conventional method to make pCCV288A (FIG. 7).

[0086] 2-4) Preparation of pCCV3251 (Example of Substitution Regarding325th Amino Acid)

[0087] DNA fragment was amplified with GeneAmpTM PCR Reagent kit(manufactured by Takara Shuzo Co., Ltd.) (1stPCR) using the mutantprimer MY-2 (100 pmol) shown by SEQ ED): No.20 and the mutant primerV325I (100 pmol) shown by SEQ ID:No. 15 and using 500 ng of pCC101obtained in Example 4 as a template DNA. The amplified DNA fragment wasdigested with restriction enzymes BstPI and XbaI, the sample waselectrophoresed with 4% agarose gel (NuSieve3:1Agarose, manufactured byTakara Shuzo Co., Ltd.), about 220 bp DNA fragments were separated andpurified using GeneClean DNA purification kit manufactured by Bio101.

[0088] On the other hand, 3 μg of pCC101 was digested with BstPI andXbaI and treated with alkaline phosphatase. Then, the BstPI-XbaIfragment (4.2 Kbp) of this pCC101 and the previously prepared andobtained BstPI-XbaI fragment (220 bp) in which mutation, had beenintroduced were ligated using a DNA ligation kit (manufactured by TakaraShuzo Co., Ltd.), and transformed into E. coli strain JM109 according tothe conventional method to make pCCV325I (FIG. 8).

[0089] 2-5) Preparation of pCCA363Term (Example of SubstitutionRegarding 363rd Amino Acid)

[0090] DNA fragment was amplified with GeneAmpTM PCR Reagent kit(manufactured by Takara Shuzo Co., Ltd.) (1stPCR) using the mutantprimer MY-2 (100 pmol) shown by SEQ ID: No.20 and the mutant primerA363term (100 pmol) shown by SEQ ID:No. 16 and using 500 ng of pCC101obtained in Example 4 as a template DNA. The resultant PCR product (150bp fragment) was purified using SUPREC-02 (manufactured by Takara ShuzoCo., Ltd.) column.

[0091] Subsequently, DNA fragment was amplified with GeneAmpTM PCRReagent kit using the mutant primer RV-D (50 pmol) shown by SEQ ID:No.19 and the 150 bp DNA fragment (50 pmol) previously purified as aprimer and using 500 ng of pCC101 as a template DNA. The amplified DNAfragment was digested with restriction enzymes BstPI and XbaI, thesample was electrophoresed with 4% agarose gel (NuSieve3:1Agarose,manufactured by Takara Shuzo Co., Ltd.), about 220 bp DNA fragments wereseparated and purified using GeneClean DNA purification kit manufacturedby Bio101.

[0092] On the other hand, 3 μg of pCC101 was digested with BstPI andXbaI and treated with alkaline phosphatase. Then, the BstPI-XbaIfragment (4.2 Kbp) of this pCC101 and the previously prepared andobtained BstPI-XbaI fragment (220 bp) in which mutation had beenintroduced were ligated using a DNA ligation kit (manufactured by TakaraShuzo Co., Ltd.), and transformed into E. coli strain JM109 according tothe conventional method to make pCCA363term (FIG. 9).

[0093] 3. Introduction of Multiple Mutation

[0094] 3-1) Preparation of pCCN43SA363Term (Example of SubstitutionRegarding 43rd and 363rd Amino Acids)

[0095] 10 μg of pCCN43S obtained in 2-1) was digested with NdeI andBpu1102I to obtain 370 bp fragment. On the other hand, 3 μg ofpCCA363term was digested with NdeI and Bpu1102I and treated withalkaline phosphatase. Then, NdeI-Bpu1102I fragment (4.2 Kbp) of thispCCA363term and the previously prepared and obtained NdeI-Bpu1102Ifragment (370 bp) were ligated using DNA ligation kit (manufactured byTakara Shuzo Co., Ltd.) and transformed into E. coli strain JM109aaccording to the conventional method to obtain the plasmidpCCN43SA363term containing the present multiple mutation gene (FIG. 10).

[0096] 3-2) Preparation of pCCT240AV288A (Example of SubstitutionRegarding 240th and 288th Amino Acids)

[0097] 10 μg of pCCT240A obtained in 2-2) was digested with ClaI andMluI to obtain 200 bp fragment. On the other hand, 3 μg of pCCV288A wasdigested with ClaI and MluI and treated with alkaline phosphatase. Then,ClaI-MluI fragment (4.4 Kbp) of this pCCV288A and the previouslyprepared and obtained ClaI-MluI fragment (200 bp) were ligated using DNAligation kit (manufactured by Takara Shuzo Co., Ltd.) and transformedinto E. coli strain JM109 according to the conventional method to obtainthe plasmid pCCT240AV 288A containing the present multiple mutation gene(FIG. 11).

Example 6 Production of the Present Esterase by TransformantMicroorganism

[0098] Total four strains of recombinant E. coli in which 3 kinds of thepresent esterase expression plasmids were introduced(JM109/pCCN43SA363term, JM109/pCCT240AV288A, JM109/pCCV325I) obtained inExample 5 and the transformant E. coli in which the wild-type esteraseexpression vector was introduced (JM109/pCC101) were inoculated on 50 mL(500 mL flask) of an LB medium (tryptone 1 (w/v) %, yeast extract 0.5(w/v) %, NaCl 0.5 (w/v) %), cultured by shaking at 37° C. and IPTG(isopropyl-β-D-thiogalactopyranoside) was added to the finalconcentration of 1 mM at logarithmic growth phase (about 2 hours afterinitiation of culturing), followed by further culturing for 4 hours.

[0099] The cells were collected by centrifugation (8000×g, 10 minutes,4° C.), and a sample prepared from the cells (having an equivalentamount to an amount of cells contained in 5 μl of culturing medium) wasanalyzed by SDS-PAGE, as a result protein was recognized as a main bandat the position corresponding to the molecular weight of the presentesterase in all the four samples.

Example 7 Purification of the Present Esterase

[0100] Four kinds of transformant E. coli which had been culturedaccording to the manner as in Example 6 were ultrasonically disrupted(20 KHz, 15 minutes, 4° C.), respectively, and centrifuged (12000×g, 30minutes, 4° C.) to obtain the supernatant. 150 ml of the resultingsupernatant was passed through a column filled with 200 ml of a negativeion-exchange resin (DEAE-Sepharose fastflow, manufactured by Pharmacia).The column was washed with 0.15M NaCl+10 mM Tris-HCl buffer (pH 7.5) andthe present esterase was eluted with 0.15-0.35M NaCl linearconcentration gradient. Measurement of the activities of the elutedfractions was performed using p-nitrophenyl acetate (pNPA) which is ageneral substrate for esterase. More particularly, 5 mM of a substratedissolved in acetonitrile was added to 1.0 ml of 10 mM phosphate buffer(pH 7.5) containing the eluted fraction, which was maintained at 37° C.and an increase in absorbance at 410 nm was measured. The fractions inwhich the esterase activity was shown were collected, and the fractionswere passed through a column filled with 200 ml of a hydrophobic resin(Butyl-Toyopearl 650S, manufactured by Toyosodakogyo). The column waswashed with 10% (w/v) (NH₄)₂SO₄+10 mM Tris-HCl buffer (pH 7.5), and thepresent esterase was eluted with 10-0% (w/v) saturated ammonium sulfatelinear concentration gradient. The fractions in which the esteraseactivity was shown were collected and adopted as a purified enzyme(hereinafter referred to as the present esterase N43SA362term,T240AV288A and V325I, and the wild-type (WT)).

Example 8 Measurement of the Thermostability of the Present Esterase

[0101] The thermal stability of four kinds of purified enzymes obtainedin Example 3 was measured according to the following procedures.

[0102] 1.0 ml of 10 mM phosphate buffer (pH 7.5) with 10 μg/ml of theabove purified enzyme added was maintained at 70° C. for 120 minutes andthe activity of the present esterase was measured. Measurement of theactivity was performed using p-nitrophenyl acetate (pNPA) which is ageneral substrate for esterase. More particularly, 5 mM substratedissolved in acetonitrile was added to the test solution aftermaintaining a temperature, maintained at 37° C. and absorbance at 410 nmwas measured. The results are shown in Table 1. A rate of the activityafter temperature maintenance at 70° C. for 120 minutes to that beforethe temperature maintenance is expressed as remaining activitypercentage and a rate of the remaining activity of the present-esterasewhen the remaining activity percentage of the wild-type esterase (WT) isregarded as 100 is shown as the remaining activity ratio (to that ofWT). TABLE 1 Remaining activity Remaining ratio activity (to that ofpercentage WT) (%) Remarks N43SA363term 173 60.0 Amino acid substitutionin which 43rd amino acid in the amino acid sequence shown by SEQ ID: No.1 is substituted with serine T240AV288A 171 59.3 Amino acid substitutionin which 240th amino acid in the amino acid sequence shown by SEQ ID:No. 1 is substituted with alanine and 288th amino acid is substitutedwith alanine V325I 163 56.6 Amino acid substitution in which 325th aminoacid in the amino acid sequence shown by SEQ ID: No. 1 is substitutedwith isoleucine Wild-type 100 34.7 esterase (WT)

[0103] As mentioned above, the present invention makes possible toprovide an esterase which may be utilized for organic synthesis reactionfor manufacturing medicaments, pesticides or intermediates thereof andis excellent in thermostability.

1 24 1 1110 DNA Chromobacterium SC-YM-1 (FERM BP-6703) CDS (1)..(1110) 1atg act ctg ttc gat ggt atc act tcg cga atc gta gat act gat cgt 48 MetThr Leu Phe Asp Gly Ile Thr Ser Arg Ile Val Asp Thr Asp Arg 1 5 10 15ctg act gtt aac atc ctg gaa cgt gcg gcc gac gac ccg cag acc ccg 96 LeuThr Val Asn Ile Leu Glu Arg Ala Ala Asp Asp Pro Gln Thr Pro 20 25 30 cccgac cgc acg gtc gtg ttc gtc cac ggg aat gtg tcc tcc gcg ctg 144 Pro AspArg Thr Val Val Phe Val His Gly Asn Val Ser Ser Ala Leu 35 40 45 ttc tggcag gag atc atg cag gac ctg ccg agc gac ctg cgc gcc atc 192 Phe Trp GlnGlu Ile Met Gln Asp Leu Pro Ser Asp Leu Arg Ala Ile 50 55 60 gcg gtc gacctg cgc ggc ttc ggc ggc tcg gag cac gcg ccg gtc gac 240 Ala Val Asp LeuArg Gly Phe Gly Gly Ser Glu His Ala Pro Val Asp 65 70 75 80 gcc acc cgcggc gtc cgc gac ttc agc gac gat ctg cac gcg acc ctc 288 Ala Thr Arg GlyVal Arg Asp Phe Ser Asp Asp Leu His Ala Thr Leu 85 90 95 gag gcg ctc gacatc ccg gtc gcg cat ctg gtc ggc tgg tcg atg ggc 336 Glu Ala Leu Asp IlePro Val Ala His Leu Val Gly Trp Ser Met Gly 100 105 110 ggc ggc gtc gtcatg cag tat gcc ctc gac cac ccg gtg ctg agc ctg 384 Gly Gly Val Val MetGln Tyr Ala Leu Asp His Pro Val Leu Ser Leu 115 120 125 acc ctg cag tcgccg gtg tcg ccc tac ggc ttc ggc ggc acc cgc cgt 432 Thr Leu Gln Ser ProVal Ser Pro Tyr Gly Phe Gly Gly Thr Arg Arg 130 135 140 gac ggc tca cgcctc acc gac gac gat gcc ggc tgc ggt ggc ggc ggt 480 Asp Gly Ser Arg LeuThr Asp Asp Asp Ala Gly Cys Gly Gly Gly Gly 145 150 155 160 gcg aac cccgac ttc atc cag cgc ctc atc gac cac gac acc tcc gac 528 Ala Asn Pro AspPhe Ile Gln Arg Leu Ile Asp His Asp Thr Ser Asp 165 170 175 gat gcg cagacc tcg ccc cgg agc gtc ttc cgc gcc ggc tac gtc gcc 576 Asp Ala Gln ThrSer Pro Arg Ser Val Phe Arg Ala Gly Tyr Val Ala 180 185 190 tcg gac tacacc acc gac cac gag gac gtg tgg gtc gaa tcg atg ctc 624 Ser Asp Tyr ThrThr Asp His Glu Asp Val Trp Val Glu Ser Met Leu 195 200 205 acc acg tccacc gcc gac gga aac tac ccc ggc gat gcg gtg ccg agc 672 Thr Thr Ser ThrAla Asp Gly Asn Tyr Pro Gly Asp Ala Val Pro Ser 210 215 220 gac aac tggccg ggc ttc gcc gcc ggc cgc cac ggc gtg ctg aac acc 720 Asp Asn Trp ProGly Phe Ala Ala Gly Arg His Gly Val Leu Asn Thr 225 230 235 240 atg gccccg cag tac ttc gat gtg tcg ggg att gtc gac ctg gcc gag 768 Met Ala ProGln Tyr Phe Asp Val Ser Gly Ile Val Asp Leu Ala Glu 245 250 255 aag cctccg atc ctg tgg atc cac ggc acc gcg gac gcg atc gtc tcc 816 Lys Pro ProIle Leu Trp Ile His Gly Thr Ala Asp Ala Ile Val Ser 260 265 270 gac gcgtcg ttc tac gac ctc aac tac ctc ggc cag ctg ggc atc gtc 864 Asp Ala SerPhe Tyr Asp Leu Asn Tyr Leu Gly Gln Leu Gly Ile Val 275 280 285 ccc ggctgg ccc ggc gaa gac gtc gcg ccc gcg cag gag atg gtg tcg 912 Pro Gly TrpPro Gly Glu Asp Val Ala Pro Ala Gln Glu Met Val Ser 290 295 300 cag acccgc gat gtc ctc ggc cgc tac gct gcg ggc ggc gga acg gtc 960 Gln Thr ArgAsp Val Leu Gly Arg Tyr Ala Ala Gly Gly Gly Thr Val 305 310 315 320 accgag gtc gcc gtc gag ggc gcg ggc cac tcc gcg cac ctg gag cgt 1008 Thr GluVal Ala Val Glu Gly Ala Gly His Ser Ala His Leu Glu Arg 325 330 335 cccgcg gtg ttc cgc cac gcg ctg ctc gag atc atc ggc tac gtc ggc 1056 Pro AlaVal Phe Arg His Ala Leu Leu Glu Ile Ile Gly Tyr Val Gly 340 345 350 gcggcg gcc gac ccc gcc ccg ccg acc gag gcg atc atc atc cgc tcc 1104 Ala AlaAla Asp Pro Ala Pro Pro Thr Glu Ala Ile Ile Ile Arg Ser 355 360 365 gccgac 1110 Ala Asp 370 2 370 PRT Chromobacterium SC-YM-1 (FERM BP-6703) 2Met Thr Leu Phe Asp Gly Ile Thr Ser Arg Ile Val Asp Thr Asp Arg 1 5 1015 Leu Thr Val Asn Ile Leu Glu Arg Ala Ala Asp Asp Pro Gln Thr Pro 20 2530 Pro Asp Arg Thr Val Val Phe Val His Gly Asn Val Ser Ser Ala Leu 35 4045 Phe Trp Gln Glu Ile Met Gln Asp Leu Pro Ser Asp Leu Arg Ala Ile 50 5560 Ala Val Asp Leu Arg Gly Phe Gly Gly Ser Glu His Ala Pro Val Asp 65 7075 80 Ala Thr Arg Gly Val Arg Asp Phe Ser Asp Asp Leu His Ala Thr Leu 8590 95 Glu Ala Leu Asp Ile Pro Val Ala His Leu Val Gly Trp Ser Met Gly100 105 110 Gly Gly Val Val Met Gln Tyr Ala Leu Asp His Pro Val Leu SerLeu 115 120 125 Thr Leu Gln Ser Pro Val Ser Pro Tyr Gly Phe Gly Gly ThrArg Arg 130 135 140 Asp Gly Ser Arg Leu Thr Asp Asp Asp Ala Gly Cys GlyGly Gly Gly 145 150 155 160 Ala Asn Pro Asp Phe Ile Gln Arg Leu Ile AspHis Asp Thr Ser Asp 165 170 175 Asp Ala Gln Thr Ser Pro Arg Ser Val PheArg Ala Gly Tyr Val Ala 180 185 190 Ser Asp Tyr Thr Thr Asp His Glu AspVal Trp Val Glu Ser Met Leu 195 200 205 Thr Thr Ser Thr Ala Asp Gly AsnTyr Pro Gly Asp Ala Val Pro Ser 210 215 220 Asp Asn Trp Pro Gly Phe AlaAla Gly Arg His Gly Val Leu Asn Thr 225 230 235 240 Met Ala Pro Gln TyrPhe Asp Val Ser Gly Ile Val Asp Leu Ala Glu 245 250 255 Lys Pro Pro IleLeu Trp Ile His Gly Thr Ala Asp Ala Ile Val Ser 260 265 270 Asp Ala SerPhe Tyr Asp Leu Asn Tyr Leu Gly Gln Leu Gly Ile Val 275 280 285 Pro GlyTrp Pro Gly Glu Asp Val Ala Pro Ala Gln Glu Met Val Ser 290 295 300 GlnThr Arg Asp Val Leu Gly Arg Tyr Ala Ala Gly Gly Gly Thr Val 305 310 315320 Thr Glu Val Ala Val Glu Gly Ala Gly His Ser Ala His Leu Glu Arg 325330 335 Pro Ala Val Phe Arg His Ala Leu Leu Glu Ile Ile Gly Tyr Val Gly340 345 350 Ala Ala Ala Asp Pro Ala Pro Pro Thr Glu Ala Ile Ile Ile ArgSer 355 360 365 Ala Asp 370 3 44 DNA Artificial Sequence modified_base(1)..(44) any n = i (inosine) 3 acnctnttcg acggnatcac ntgncgnatcgtngacacng accg 44 4 44 DNA Artificial Sequence modified_base (1)..(44)any n = i (inosine) 4 acnctnttcg acggnatcac ntcncgnatc gtngacacng accg44 5 20 DNA Artificial Sequence Description of Artificial SequenceOligonucleotide LP-1 5 aattcttttt taataaaatc 20 6 26 DNA ArtificialSequence Description of Artificial Sequence Oligonucleotide LP-2 6ttttcctcct gattttatta aaaaag 26 7 30 DNA Artificial Sequence Descriptionof Artificial Sequence Oligonucleotide ES-3 7 ggtatcactt cgcgaatcgtagatactgat 30 8 45 DNA Artificial Sequence Description of ArtificialSequence Oligonucleotide ES-4 8 ggccgcacgt tccaggatgt taacagtcagacgatcagta tctac 45 9 29 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide ES-5 9 cgtctgactg ttaacatcctggaacgtgc 29 10 37 DNA Artificial Sequence Description of ArtificialSequence Oligonucleotide ES-6 10 gattcgcgaa gtgataccat cgaacagagtcatatgt 37 11 30 DNA Artificial Sequence Description of ArtificialSequence Oligonucleotide ES-7 11 aggaggaaaa aacatatgac tctgttcgat 30 1230 DNA Artificial Sequence Description of Artificial SequenceOligonucleotide N43S 12 cagcgcggag gacacagacc cgtggacgaa 30 13 30 DNAArtificial Sequence Description of Artificial Sequence OligonucleotideT240A 13 ggcgtgctga acgccatggc cccgcagtac 30 14 30 DNA ArtificialSequence Description of Artificial Sequence Oligonucleotide V288A 14cagctgggca tcgcccccgg ctggcccggc 30 15 35 DNA Artificial SequenceDescription of Artificial Sequence Oligonucleotide V325I 15 ggcggaacggtcaccgaggt cgccatcgag ggcgc 35 16 30 DNA Artificial Sequence Descriptionof Artificial Sequence Oligonucleotide A363term 16 ccgccgaccg agtgaatctaaatccgctcc 30 17 28 DNA Artificial Sequence Description of ArtificialSequence Oligonucleotide RV-G 17 gaccatgatt acgaattctt ttttaata 28 18 30DNA Artificial Sequence Description of Artificial SequenceOligonucleotide RC-C 18 gaccacccgg tgctgagcct gaccctgcag 30 19 30 DNAArtificial Sequence Description of Artificial Sequence OligonucleotideRV-D 19 ggcggaacgg tcaccgaggt cgccgtcgag 30 20 29 DNA ArtificialSequence Description of Artificial Sequence Oligonucleotide MY-2 20cgacggccag tgccaagctt gcatgccgc 29 21 30 DNA Artificial SequenceDescription of Artificial Sequence Oligonucleotide MY-3 21 gtcgatgaggcgctggatga agtcggggtt 30 22 30 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide MY-6 22 ctcgacggcg acctcggtgaccgttccgcc 30 23 109 DNA Artificial Sequence Description of ArtificialSequence double stranded DNA fragment (SD) 23 aattcttttt taataaaatcaggaggaaaa aacatatgac tctgttcgat ggtatcactt 60 cgcgaatcgt agatactgatcgtctgactg ttaacatcct ggaacgtgc 109 24 109 DNA Artificial SequenceDescription of Artificial Sequence double stranded DNA fragment (SD) 24gaaaaaatta ttttagtcct ccttttttgt atactgagac aagctaccat agtgaagcgc 60ttagcatcta tgactagcag actgacaatt gtaggacctt gcacgccgg 109

1. An esterase which is characterized in that it has at least a partialamino acid sequence necessary for expressing the thermostable esteraseactivity among the amino acid sequence shown by SEQ ID: No.1 having anyone of the following amino acid substitutions: (1) amino acidsubstitution where 325th amino acid in the amino acid sequence shown bySEQ ID: No.1 is substituted with isoleucine, (2) amino acid substitutionwhere 240th amino acid in the amino acid sequence shown by SEQ ID: No.1is substituted with alanine, and 288th amino acid is substituted withalanine, (3) amino acid substitution where 43rd amino acid in the aminoacid sequence shown by SEQ ID: No.1 is substituted with serine.
 2. Anesterase which is characterized in that it has at least a partial aminoacid sequence necessary for expressing the thermostable esteraseactivity among the amino acid sequence shown by SEQ ID: No.1 havingamino acid substitution where 325th amino acid in the amino acidsequence shown by SEQ ID: No.1 is substituted with isoleucine.
 3. Anesterase which is characterized in that it has at least a partial aminoacid sequence necessary for expressing the thermostable esteraseactivity among the amino acid sequence shown by SEQ ID: No.1 havingamino acid substitution where 240th amino acid in the amino acidsequence shown by SEQ ID: No.1 is substituted with alanine, and 288thamino acid is substituted with alanine.
 4. An esterase which ischaracterized in that it has at least a partial amino acid sequencenecessary for expressing the thermostable esterase activity among theamino acid sequence shown by SEQ ID: No.1 having amino acid substitutionwhere 43rd amino acid in the amino acid sequence shown by SEQ ID: No.1is substituted with serine.
 5. A gene which is characterized in that itencodes the esterase of claims 1 to
 4. 6. A plasmid which ischaracterized in that it contains the gene of claims
 5. 7. Amicroorganism which is characterized in that it contains the plasmid ofclaim
 6. 8. A process for producing an esterase, which is characterizedby comprising culturing the microorganism of claim 4 and, thereby,allowing the microorganism to produce an esterase having at least apartial amino acid sequence necessary for expressing the thermostableesterase activity among the amino acid sequence shown by SEQ ID: No.1having any one of the following amino acid substitutions: (1) amino acidsubstitution where 325th amino acid in the amino acid sequence shown bySEQ ID: No.1 is substituted with isoleucine, (2) amino acid substitutionwhere 240th amino acid in the amino acid sequence shown by SEQ ID: No.1is substituted with alanine and 288th amino acid is substituted withalanine, (3) amino acid substitution where 43rd amino acid in the aminoacid sequence shown by SEQ ID: No.1 is substituted with serine.